1. Technical Field
The invention relates to the field of biopharmaceutical process development with eukaryotic cells that produce recombinant protein of interest. In particular, the invention relates to a method of optimising the management of the process, which comprises using an Na2CO3 and NaHCO3 free or reduced medium in conjunction with a pCO2 regulation.
2. Background
Biopharmaceutical process development is confronted with the requirement of providing ever higher titres for the production of therapeutic proteins, particularly antibodies for clinical (toxicological) studies with tight deadlines, or for supplying the market.
Within the scope of these time constraints, expression systems have to be designed, stable production cell clones have to be generated and selected for production (e.g. CHO cells, hybridomas, BHK or NSO cells), scalable biopharmaceutical production processes have to be designed, comprising media optimisation and process control. All this has to be addressed so that the specific productivity (=specific product formation rate) and the titres achieved (product yield) are maximised, to enable the processes to be run robustly and reproducibly in plants of different sizes. In recent years enormous increases in product titres have been achieved in recombinant eukaryotic cell systems. Thus, for example, product concentrations of more than 5 g of immunoglobulin/L in Chinese Hamster Ovary (CHO) cells have been achieved. Progress in molecular biology and cell biology includes genetic cell line development and changes, development of media, and “in-process” control strategies such as the addition of nutrients (“feed”) have made this possible, inter alia. However, the productivities of eukaryotic cell systems, particularly mammalian cell systems, do not approach those of prokaryotic cell systems and therefore the further optimisation of, in particular, process control during fermentation and the optimisation of the fermentation medium is still a requirement.
One particular requirement is the control of the oxidative metabolism.
All cells produce CO2 within the scope of oxidative metabolism and require HCO3− for a variety of related transport processes. Elevated CO2 contents, which may occur, for example, when there is strong growth, lower the pH, and this can be neutralised by an increased sodium hydrogen carbonate content. Thus, sodium hydrogen carbonate is both a buffer substance and an essential nutrient. Sodium hydrogen carbonate is described as an essential nutrient in, among others, the textbook by Lindl, Toni; Gstraunthaler Gerhard (2002): Zell- und Gewebekultur [Cell and Tissue Culture], 5th edition, Spektrum Akademischer Verlag Heidelberg, p. 93, Point 4.4.3, or in Ling, C. T. et al (1968): Chemically characterized concentrated corodies for continuous cell culture (The 7C's culture media), in Experimental Cell Research Nr. 52, p. 469-489.
Moreover, hydrogen carbonate plays a part in the citric acid cycle, in the pH regulation of the whole body as well as individual cells, and regulation of their volume. The membranes of mammalian cells contain transport proteins for hydrogen carbonate to assist with transmembrane transport. Whereas CO2 is capable of penetrating through the lipid bilayer by diffusion, HCO3− is charged and can only pass through the membrane with the aid of specific transport proteins. On the basis of the acid-base properties of CO2/HCO3−, the expulsion of CO2 from the cell results in an alkalisation of the cell, whereas the efflux of HCO3− acidifies the cell (Casey Joseph. R. (2006): Why bicarbonate? in Biochem. Cell Biol. No. 84, S. 930-939).